A biologically active rhIGF-1 fusion accumulated intransgenic rice seeds can reduce blood glucose in diabeticmice via oral delivery

Tingting Xie a, Qingchuan Qiu a, Wei Zhang b, Tingting Ning a, Wei Yang c, Congyi Zheng c,Chuan Wang a, Yingguo Zhu a, Daichang Yang a,*aCenter of Engineering and Research of the Ministry of Education for Plant Biotechnology and Germplasm Utilization,

Department of Genetics, College of Life Sciences, Wuhan University, Wuhan 430072, PR Chinab School of Medicine, Wuhan University, Wuhan 430072, PR ChinacState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 0

a r t i c l e i n f o

Article history:

Received 27 March 2008

Received in revised form

30 June 2008

Accepted 7 July 2008

Published on line 29 July 2008

Keywords:

Human insulin-like growth factor 1

Transgenic rice seeds

Blood glucose

Hyperglycemia

Diabetes mellitus

a b s t r a c t

Human insulin-like growth factor 1(hIGF-1) is essential for cell proliferation and used

therapeutically in treating various diseases including diabetes mellitus. Here, we present

that a recombinant hIGF-1(rhIGF-1) was expressed fused with the C-terminus of a rice

luminal binding protein and accumulated highly in rice seeds, reaching 6.8 � 0.5% of total

seed protein. The rhIGF-1 fusion was demonstrated to possess biological activity to stimu-

late cell proliferation. Importantly, the unprocessed transgenic seeds could significantly

increase plasma rhIGF-1 level and reduce blood glucose of diabetic mice via oral delivery.

Further studies suggested that transgenic seeds reduced blood glucose of diabetic mice by

enhancing islet cells survival and increasing insulin secretion rather than increasing insulin

sensitivity. These results indicated the potential of the novel fusion expression system in

production and oral delivery of biologically active small peptides for diseases.

# 2008 Elsevier Inc. All rights reserved.

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1. Introduction

Human insulin-like growth factor 1 (hIGF-1), a single-chain

small peptide of 70 amino acids, is a predominant growth

factor in regulating growth, survival, and metabolism [12].

Clinically, hIGF-1 is used to effectively treat patients with

growth hormone (GH) receptor deficiency, GH insensitivity

syndrome, hIGF-1 gene deletion or defects in GH signal

transduction pathways [19]. Furthermore, hIGF-1 can be

effectively used in treating patients with type 1 and type 2

diabetes mellitus, or severe insulin resistance syndromes [5].

Increasing data indicate that the potential therapeutic

applications of hIGF-1 are extremely board and encouraging,

* Corresponding author. Tel.: +86 27 68754680; fax: +86 27 68754680.E-mail address: dyang@whu.edu.cn (D. Yang).

0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2008.07.014

however, hIGF-1 has not been attempted for those applica-

tions yet, in part due to the shortage of adequate supplies

[19].

To satisfy market demand, several different host-vector

systems to produce recombinant human IGF-1 (rhIGF-1) have

been exploited, including E. coli [11], yeast [26], transgenic

rabbits [4,37], and transgenic plants [16]. Although progress in

those expression systems has been achieved, several limita-

tions hinder the maximum output of biologically active and

safe therapeutic agents. Currently, growing evidence show

that cereal seeds could be an ideal production platform for

plant-made pharmaceuticals (PMP) because they have tre-

mendous advantages such as high expression level, stable

.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 0 1863

accumulation, large biomass, low production costs and easy

oral administration [21].

In this study, we developed transgenic rice plants that could

accumulate rhIGF-1 fusion in seeds. High accumulation was

achievedbyfusingrhIGF-1withtheC-terminus ofan ERluminal

binding protein (BipC). The rhIGF-1 fusion was accumulated to

6.8� 0.5% of total seed protein, which was equivalent to

136� 10 mg per seed. A functional analysis indicated that

rhIGF-1 fusion could stimulate the proliferation of human

breast carcinoma cell line MCF-7 in vitro. Animal test showed

that oral administration of unprocessed transgenic seeds in

diabetic mice could significantly increase plasma rhIGF-1 level

and reduce blood glucose. Glucose and insulin tolerance tests

indicated that transgenic seeds lower blood glucose by

increasing insulin secretion rather than insulin sensitivity.

Further histological observation suggested that rhIGF-1 stimu-

late insulinsecretion by enhancing islet survival. This is the first

report to directly use oral administration of rice seed-based

rhIGF-1 fusion to effectively treat diabetes in mice.

2. Materials and methods

2.1. Plasmid construction

All enzymes used in the study were purchased from New

England Biolabs. DNA sequences coding for hIGF-1 (Genbank

Fig. 1 – Expression of rhIGF-1 fusion in the transgenic rice. (A) S

pOsPMP26 plasmid. The DNA fragments coding for the BipC and

glutelin Gt13a promoter, in-frame fused with Gt13a correspondin

(B) Expression analysis of rhIGF-1 fusion by PAGE and Western

three independent transgenic lines and non-transgenic TP309 s

detected with anti-hIGF-1 antibody. (C) Southern blot analysis o

young leaves was digested by HindIII, EcoRI and both of HindII

probe. A rice cultivar TP309 served as a control. (D) Expression s

extracts from homozygous seeds of line 26-13 fromT1 to T3 gen

by 12% SDS-PAGE and immuno-detected with anti-human IGF-I

corresponding to the bands in Western blot of transgenic lines

accession No. CAA01955) and 256 amino acids from C-

terminus of rice Bip (Genbank accession No. AAB63469) were

synthesized by Heron Blue Biotechnology Inc (Bothell, WA)

using rice-preferred genetic codons. To construct a vector of

pBipC:IGF-1, the DNA sequence of hIGF-1 was amplified

from pUC18-higf plasmid using a forward primer: 50-

catgccatggGGCCCGGAGACCCTCTGC-30 and a reverse pri-

mer: 50-ATTCGGCTCCGCTCGAGTTC-30. The PCR product

was inserted between Gt13a promote/Gt13a corresponding

signal peptide and Nos terminator, which contained in the

pOsPMP02 vector [15], by the NcoI and XhoI sites. Then,

the bipc fragment was amplified from pUC18-bipc plasmid

using a forward primer: 50-ctaggatatcCTCTCCGGCGAGGGC-

30 and a reverse primer: 50-TTAGGCACCCCAGGCTTTACAC-

30. The PCR product was in-frame cloned between the

Gt13a signal peptide and igf-1 gene by the NaeI and NcoI

sites. The resulting construct was designated as pOsPMP26

(Fig. 1A).

2.2. Transformation and plant regeneration

pOsPMP26 was co-transformed with plasmid pOsPMP05

containing a selective marker gene [15] through micropro-

jectile-mediated transformation using callus regenerated

from rice cultivar TP309 as described previously [34].

Transgenic plants were grown in a greenhouse at Wuhan

University.

chematic representation of the restriction map of the

hIGF-1 were under the control of a rice storage protein

g signal peptide, and terminated by Nos terminator (Nos T).

blot. Total proteins extracts from homozygous seeds of

eeds were separated by 12% SDS-PAGE and immuno-

f the transgenic line 26-13. Genomic DNA isolated from

I and EcoRI, and then hybridized with a DIG-labelled igf

tability analysis of the transgenic line 26-13. Total proteins

erations and non-transgenic TP309 seeds were separated

antibody. Arrow indicates a distinct �38 kDa protein bands

, but it is absent in TP309 in panel B and D.

Table 1 – Treatment of diabetic mice

Day 0–3 Day 3–6 Day 6–9 Day 9–12

Group I Feed A Feed A Feed B Feed B

Group II Feed C Feed A Feed A Feed A

Group III Feed B Feed B Feed B Feed B

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2.3. Southern analysis

Genomic DNA was isolated from the young rice leaves as

described by Dellaporta et al. [6]. About 5 mg of the genomic

DNA was digested by HindIII, EcoRI and both of HindIII and

EcoRI. After being separated by 1% agarose gel, the DNA was

blotted onto a nylon membrane following the manufacturer’s

instructions (Millipore, Billerica, MA), and then probed with

the igf-1 gene using DIG High Prime DNA Labeling and

Detection Starter Kit I (Roche Diagnostics, Inc., Mannheim,

Germany).

2.4. Protein extraction, detection, and quantification

Total protein extracts from seeds and other tissues were

prepared by grinding the tissues under liquid nitrogen and

extracting total protein with protein extraction buffer (66 mM

Tris, pH 6.8, 2% SDS, 1 mM DTT). After being separated by 12%

SDS-PAGE and transferred to PVDF membranes (Millipore),

rhIGF-1 fusion was detected by anti-hIGF-1 monoclonal

antibody (R&D system, Wiesbaden, Germany) followed by

anti-mouse IgG, coupled to alkaline phosphatase. For rhIGF-1

fusion quantification, 0.2 g seed was ground in 10 ml total

protein extraction buffer. The concentration of rhIGF-1 fusion

was determined by titration using a known concentration of

�45 kDa egg albumin (Sigma, St-Louis, MO).

Soluble protein extracts were prepared from transgenic

immature seeds harvested at 28 days after pollination (DAP)

with 1 � PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM

Na2HPO4, 2 mM KH2PO4, pH 7.4).

2.5. Expression profile of rhIGF-1 fusion duringseed development

Total seed protein concentration of the extracts from

immature seeds harvested at 7, 14, 21, and 28 DAP was

determined by using the Bio-Rad Protein Assay system

(BioRad, Hercules, CA). Thirty micrograms of total protein at

each stage were loaded and separated by 12% PAGE. Western

blot and quantification were performed as described above.

2.6. Transmission and immuno-electron microscopy

Immature seeds were harvested at 10–14 DAP. Fixation,

sectioning and immuno-electronic microscopic observations

followed the procedure as previously described [33].

2.7. Cell proliferation assays

Human breast carcinoma cell line MCF-7 was obtained from

the China Center for Type Culture Collection. MCF-7 cells were

cultured in RPMI1640 medium (Sigma) supplemented with 10%

fetal bovine serum (FBS, Promega Corp., Madison, WI). Then,

the cells were plated and cultured as described by Xia et al.

[29]. After the cell attachment to the plate wall and serum

starvation treatment, an aliquot of 0.1 ml serum-free medium

(SFM) was added to each of the wells containing one of the

following components: 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/

ml, 6.25 ng/ml of E. coli-derived rhIGF-1 (R&D system), 50 ml/ml

soluble protein extracts from 28 DAP transgenic immature

seeds (SPETS), and 28 DAP non-transformed immature seeds

(SPENTS), respectively. SFM and a complete medium (SFM

with 10% FBS) were used as a negative and a positive control,

respectively. Cell viability was monitored after 24 h of culture

by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium

bromide (MTT, Sigma) assay [29]. Background was eliminated

by subtracting the optical density (OD570) of the negative

control. A reading of OD570 in the complete medium was

standardized as 100%.

2.8. ELISA

A human IGF-I DuoSet ELISA kit was purchased from R&D

System. The ELISA protocol was operated according to the

manufacturer’s instruction.

2.9. Mice feeding experiments

Six-week-old (26 � 3 g) male KunMing (KM) mice were

obtained from the Experimental Animal Center of Wuhan

University. Blood glucose levels were measured using the

OneTouch1 Ultra1 Meter and corresponding test strips

(Lifescan; Johnson & Johnson, Milpitas, CA). The diabetic mice

were induced by streptozotocin (STZ, Sigma) as described by

George et al. [7]. After 8 h of starvation, the mice with 200 mg/

ml or higher blood glucose were deemed to be diabetic mice.

Those diabetic mice were randomly divided into three groups

(n = 6 per group). The grouping date was defined as day 0. The

treatment of each group showed in Table 1. Feeds A, B and C

contained 40% transgenic seeds, 40% non-transgenic seeds,

and 10% transgenic seeds, respectively. Blood glucose level

was recorded at days 0, 3, 6, 9, and 12. Food intake and water

intake were recorded daily. All procedures were performed

under an approved protocol in compliance with guidelines set

by the Experimental Animal Center of Wuhan University.

2.10. Measure of rhIGF-1 protein levels in the plasma

Experimental blood samples were harvested in diabetic mice

at 9 days after fed by Feed A or Feed B. Plasma were extracted

from blood samples by centrifugation at 3000 rpm for 10 min

and then pre-treated by acid-ethanol cryo-precipitation

method to separate IGF-1 from binding proteins as previously

described [3]. Plasma rhIGF-1 levels were measured by human

IGF-I DuoSet ELISA kit (R&D system, Wiesbaden, Germany) as

described by the manufacturer’s instruction.

2.11. Glucose and insulin tolerance tests

For glucose and insulin tolerance tests, overnight-fasted

diabetic mice were given intraperitoneal glucose (2 mg/g body

weight) or insulin (Humulin, 0.75milli international units/g),

respectively. Tail blood was collected before (time 0) and at

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 0 1865

indicated times (30 min, 120 min) after injection for measure-

ment of glucose.

2.12. Histology

Pancreas tissue were fixed in 10% formaldehyde, embedded in

paraffin, sectioned and stained with hematoxylin and eosin

(H&E). Microscopy observations were performed with a

Vanox-S microscope (Olympus, Japan). Image acquisition

was carried out using a digital camera (QImaging, Canada).

The islets in pancreas were quantified using Image J analysis

Fig. 2 – Expression pattern analysis of the rhIGF-1 fusion in the tra

1 fusion. Total proteins was extracted from roots (R), stems (St), le

26-13 and separated by 12% SDS-PAGE and immuno-detected w

expressed in seeds, but not in other tissues. (B) The expression p

microliters total proteins extract from 7, 14, 21, 28 DAP immatur

Western blot. (C) Electron-microscopic (EM) observation of the de

and two types of normal protein body (PB I and PB II) were found i

endosperm from transgenic line 26-13. (E) Immuno-EM photograp

rhIGF-1 fusion was localized in the ER lumen and the PB I surfac

endosperm from transgenic line 26-13. BipC-IGF was localized in

system. Biomass and area of islets in Feed B group were

standardized as 1.0, respectively.

3. Results

3.1. Generation and characterization of the transgenicrice plants

A total of 16 independent transgenic plants were obtained.

Nine of those were fertile. For screening of the transgenic lines

nsgenic line 26-13. (A) Tissue specificity expression of rhIGF-

aves (L), inflorescence (Inf) and seeds (Se) of transgenic line

ith anti-hIGF-1 antibody. rhIGF-1 fusion was specifically

rofile of rhIGF-1 fusion during seeds development. Thirty

e seeds and mature seeds were analyzed by the PAGE and

veloping endosperm from non-transformed TP309. The ER

n the endosperm cells. (D) EM observation of the developing

hs of the developing endosperm from transgenic line 26-13.

e (arrows). (F) Immuno-EM observation of the developing

the inner PB II (arrows).

Fig. 3 – The effects of rhIGF-1 fusion on MCF-7 cell growth.

MCF-7 cells were grown in serum-free medium (SFM) with

various concentration of E. coli-derived rhIGF-1, 50 ml/ml

soluble protein extracts from 28 DAP rhIGF-I transgenic

rice seeds (SPETS) and 50 ml/ml soluble protein extracts

from 28 DAP non-transformed seeds (SPENTS),

respectively. SFM and a complete medium (SFM with 10%

FBS) were used as a negative and a positive control,

respectively. The background was eliminated by

subtracting the optical density (OD570) of the negative

control. A reading of OD570 in the complete medium was

standardized as 100%. **p < 0.001 and *p < 0.01 vs. cells in

SFM (negative control).

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 01866

highly expressing rhIGF-1 fusion, both PAGE and Western

analysis were carried out. A distinct protein band about 38 kDa

was easily seen in the transgenic seeds in PAGE stained with

Coomassie Blue, whereas it was absent in non-transformed

TP309 seeds (Fig. 1B). The distinct protein band was further

confirmed to be rhIGF-1 fusion by Western blot using an

antiserum against rhIGF-1 (Fig. 1B). The transgenic line 26-13

was one of the highest expression transgenic lines (6.8 � 0.5%

of total seed protein, Supplemental 1) and was advanced to the

next generation for further experiment.

First, the insertion locus and copy numbers of transgenic

line 26-13 were determined. For T1 seeds, 39 expression to 11

non-expression fits to a 3:1 (p = 0.62) segregation, indicating

that line 26-13 contains a single insertion in the rice genome.

The copy numbers in 26-13 line were determined by Southern

blot analysis. As shown in Fig. 1C, at least two copies of the

transgene existed in the rice genome. So we concluded that

this transgenic (26-13) line had a single insertion with 2–3

copies.

To determine rhIGF-1 fusion expression stability during

three generations, we examined the expression level of the

three generations of 26-13 line. The results showed that the

specific rhIGF-1 fusion band in 26-13 line had similar intensity

through T1–T3 generations in PAGE and Western analysis

(Fig. 1D), indicating that the expression level of rhIGF-1 fusion

was stable, at least through three generations.

3.2. Expression analysis of rhIGF-1 fusion

To determine the expression specificity of rhIGF-1 fusion

under control of a Gt13a promoter, total protein was extracted

from the root, leaf, stem, inflorescence, and mature seeds of

transgenic line 26-13 and immuno-blotted with antiserum

against hIGF-1. The results showed that rhIGF-1 fusion was

detected in seeds only, but not in root, leaf, stem and

inflorescence (Fig. 2A). This indicated that rhIGF-1 fusion

expression was seed-specific.

The fusion protein expression profile during seed devel-

opment was monitored at 7, 14, 21, and 28 DAP in immature

seeds and mature seeds in transgenic line 26-13. Accumula-

tion of rhIGF-1 fusion began at 7 DAP, then increased

dramatically between 14 and 28 DAP, and then peaked at 28

DAP, reaching the highest level of �9.0% of total seed protein.

The contents of rhIGF-1 fusion decreased a little during seed

maturation and stayed at�6.8% of total seed protein in mature

seeds (Fig. 2B). This expression pattern is similar to human

lysozyme expression in rice endosperm under control of a Gt1

promoter [33].

To figure out subcellular localization of rhIGF-1 fusion in

transgenic endosperm cells, transmission and immuno-

electron microscopy were carried out. Protein body II (PB II)

turned to be a spherical shape in the transgenic endosperm

cells compared with an irregular shape in the non-transgenic

endosperm cells, whereas protein body I (PB I) seemed to be

unaffected (Fig. 2C and D). Further results of subcellular

localization showed that rhIGF-1 fusion was clearly detected

in the endoplasmic reticulum (ER) lumen and protein body I

surface in the transgenic endosperm cells (Fig. 2E). It can be

explained that the ER retention signal HDEL in the C-terminus

of Bip can retain the fusion in ER and ER-derived PB I. In

addition, much more immune colloidal gold could also be

found to localize in PB II (Fig. 2F). It may be theoretically

explained that Bip protein could be transported out of the ER-

Golgi by saturating the HDEL receptor [17].

3.3. The biological activity of rhIGF-1 fusion incell culture assay

To investigate whether the rice seed-derived rhIGF-1 fusion is

biologically active in vitro, cell proliferation assays were

conducted by supplement with 28 DAP SPETS in MCF-7 cell

culture. First of all, we detected the concentration of the

transgenic rice-derived rhIGF-1 in SPETS by sandwich ELISA,

which was 2.09 � 0.4 mg/ml. Then, the effect of E. coli-derived

rhIGF-1(ErhIGF-1) on MCF-7 cell proliferation was evaluated.

ErhIGF-1 obviously stimulated the proliferation of MCF-7 cells

in a dose-dependent manner (Fig. 3). When 50 ml/ml 28 DAP

SPETS were added to MCF-7 medium (final concentration of

105 � 20 ng/ml rice-derived rhIGF-1), cell relative viability

reached 75.37 � 9.06%, which is more than the cell relative

viability affected by 100 ng/ml ErhIGF-1. On the other hand,

the cell relative viability was only 18.16 � 6.94% when the

same amount of 28 DAP SPENTS was added to MCF-7 medium,

which is lower than that of 6.25 ng/ml ErhIGF-1 (Fig. 3). The

results demonstrated that rhIGF-1 fusion expressed in rice

endosperm possesses high biological activity in cell prolifera-

tion.

3.4. The effect of rhIGF-1 fusion on diabetic mice

To assess the function in vivo of rhIGF-1 fusion, an oral

administration experiment was conducted by using the STZ-

induced diabetic KM mice. Blood glucose levels of the diabetic

mice were decreased rapidly and significantly from

329 � 77 mg/dl to 88 � 12 mg/dl (p < 0.001) at day 3 after oral

administration of Feed A (Fig. 4A). Blood glucose levels were

Fig. 4 – The biological effects of rhIGF-1 fusion by oral

delivery on diabetic mice. The STZ-induced diabetic mice

were random divided into three groups: group I (blank

circle), group II (solid circle) and group III (blank rectangle).

Mice in each group were fed with different feeds as

described in Section 2 (Table 1). (A) The effects of rhIGF-1

fusion on blood glucose. (B) The effects of rhIGF-1 fusion

on food intake of diabetic mice. (C) The effects of rhIGF-1

fusion on water intake of diabetic mice. **p < 0.01 and*p < 0.05 vs. group III.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 0 1867

stabilized at 81 � 37 mg/dl at day 6 when continuing oral

administration of Feed A. However, blood glucose levels

immediately returned to 372 � 44 mg/dl at day 9 and kept

increasing at day 12 when Feed A was replaced by Feed B.

Fig. 5 – Plasma rhIGF-1 level and glucose/insulin tolerance tests

was determined in Feed A group (white bars) and Feed B group

glucose was measured before and after i.p 2 mg/g glucose admin

after i.p. 0.75 mIU/g insulin administration (n = 6).

When Feed C was used instead of Feed A, blood glucose levels

of diabetic mice decreased from 329 � 88 mg/dl to

215 � 96 mg/dl (p < 0.001) at day 3. Similarly, it was observed

that blood glucose levels decreased dramatically to

85 � 45 mg/dl and stayed at low levels for the next 6 days

when the Feed C was replaced by Feed A. In contrast, blood

glucose levels kept rising from 291 � 77 mg/dl to 558 � 83 mg/

dl throughout 12 days in the control group fed with Feed B.

Meanwhile, food and water intakes corresponded to the

fluctuation in blood glucose levels in all treatments (Fig. 4B and

C), indicating that diabetic symptoms were alleviated with a

reduction in blood glucose levels.

3.5. Mechanisms of the decreased blood glucose in thetransgenic seeds-fed mice

To elucidate the mechanism of decreased blood glucose in the

transgenic seeds-fed mice, content of plasma rhIGF-1 was

measured. In this study, two group diabetic mice were fed by

Feed A (Feed A group, n = 17) and Feed B (Feed B group, n = 17)

for 9 days, respectively. Similar to the results of previous

study, blood glucose of Feed A group decreased significantly

compared with Feed B group (101 � 28 mg/dl vs. 280 � 100 mg/

dl, p < 0.001). As shown in Fig. 5A, plasma rhIGF-1 level of Feed

A group was significantly higher than the baseline measured

in Feed B group (7.1 � 3.1 ng/ml vs. 3.0 � 0.1 ng/ml, p < 0.05).

The results suggested that rhIGF-1 fusion in transgenic seeds

could be absorbed into plasma via digestive system. It was

implied that blood glucose in diabetic mice could be decreased

by enhancing rhIGF-1 level in plasma.

To confirm whether the beneficial effect of rhIGF-1 on

blood glucose was caused by increased insulin secretion and/

or insulin sensitivity, glucose and insulin tolerance tests were

performed. Glucose tolerance test showed glucose levels in

Feed A group were significantly lower than in Feed B group at

all time points after glucose administration (Fig. 5B). Insulin

tolerance test showed that blood glucose response to insulin

of Feed A group was similar to Feed B group (Fig. 5C). It

suggested that insulin sensitivity was not influenced by

increased rhIGF-1 in plasma. These results hinted that the

decrease of blood glucose in the transgenic seeds-fed mice

might be caused by increase of insulin secretion rather than

insulin sensitivity.

in diabetic mice fed with Feed A/Feed B. (A) Plasma rhIGF-1

(black bars) by ELISA. *p < 0.05 vs. Feed B group. (B) Blood

istration (n = 6). (C) Blood glucose was measured before and

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 01868

To further understand mechanism of increasing

insulin secretion in transgenic seeds-fed mice, histology

of pancreas was performed. As shown in Fig. 6A, B and

E, islet biomass in Feed A group was 2.13 times more

( p < 0.01, n = 5) than that in Feed B group. Simultaneously,

islet size in Feed A group was 1.64 times larger ( p < 0.01,

n = 5) than that in Feed B group (Fig. 6C, D and F). The

results indicated that insulin secretion might be

Fig. 6 – Histological observation of pancreas in diabetic mice fed

with Feed A, T40. (B) Pancreas of diabetic mice fed with Feed B, T

T400. (D) High magnification of the boxed region of panel (B), T

and Feed B group (black bars). Islet biomass was determined by

section (n = 6). Islet biomass in Feed B group was standardized a

Feed B group (black bars). Average islet area in each pancreas s

Average islet area in Feed B group was standardized as 1.0. Arr

enhanced by increase of islet survival in transgenic seeds-

fed mice.

4. Discussion

Direct production of small peptides is difficult for most current

expression systems in either eukaryotic or prokaryotic cells

with Feed A and Feed B. (A) Pancreas of diabetic mice fed

40. (C) High magnification of the boxed region of panel (A),

400. (E) Relative islet biomass in Feed A group (white bars)

counting the number of islets per unit area of pancreas

s 1.0. (F) Relative islet size in Feed A group (white bars) and

ection was calculated by Image J analysis system (n = 6).

ows show islets in pancreas. **p < 0.001 vs. Feed B group.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 6 2 – 1 8 7 0 1869

[16,20,36]. This problem has in general been resolved by

expressing small peptides with a fusion protein, such as fused

with seed storage proteins or inserted into the variable regions

of common seed storage proteins [22,23,35]. Here, we devel-

oped a novel non-storage protein fusion expression system to

highly accumulate rhIGF-1 in transgenic rice seeds. The rhIGF-

1 fusion can be clearly seen in PAGE stained with Coomassie

Blue and accounted for 6.8 � 0.5% of the total seed protein. As

expected, the expression of rhIGF-1 fusion was seed-specific

and stable.

Diabetes, one of the highest risk factors leading to other

health problems, is a leading cause of death in the worldwide.

Treatment of diabetes involves lowering the levels of known

risk factors that damage blood vessels, especially blood

glucose. Insulin has been used effectively to control the blood

glucose of diabetics. Since IGF-1 and insulin shared a single

IGF-1/insulin precursor, IGF-1 has retained some insulin-like

properties [5]. As a candidate drug for diabetes, the therapeutic

value of hIGF-1 has been evaluated in diabetes. Clinical trials

have shown that postprandial glucose disposal is partly

dependent upon IGF-1 concentrations and that administering

IGF-1 to patients with either severe insulin resistance or type 2

diabetes results in improved postprandial glucose usage [5].

Also, larger trials of hIGF-1 administration to type 1 diabetes

have shown a consistent maintenance of reduced insulin

requirements over 4–8-week periods [5]. Recent studies show

that hIGF-1 lowers blood glucose by enhancing insulin

sensitivity in both type 1 diabetes and type 2 diabetes

[18,31]. Other studies suggest that hIGF-1 lowers blood glucose

level by enhancing b cell regeneration, preventing b cell

apoptosis and increasing insulin secretion [2,7,8]. In our study,

we find that the transgenic rice seed-derived rhIGF-1 fusion

could efficiently reduce blood glucose of STZ-induced diabetic

mice. Glucose and insulin tolerance tests indicate that

transgenic seeds lower blood glucose by increasing insulin

secretion rather than insulin sensitivity. Further studies

demonstrate that rhIGF-1 stimulate insulin secretion by

enhancing islet survival. The findings are consistent with

the study of b cell replication by IGF-1 treatment in vitro [10]

and regeneration pancreatic islets by local expression of IGF-1

in b cell [7,8].

Currently, oral delivery of peptides and proteins remains

an attractive alternative to subcutaneous injection. It is so far

one of the easiest and most widely used routes of drug delivery

especially when repeated or routine dosing is necessary,

besides it is less invasive and cheaper [14,30]. However, it is

thought that oral delivery of small peptides or proteins is

generally not feasible because of its pre-systemic enzymatic

degradation and poor penetration of the intestinal membrane

[9,13]. Nevertheless, in this study, we demonstrated that

rhIGF-1 from transgenic rice seeds could increase the plasma

rhIGF-1 level and efficiently reduce blood glucose of diabetic

mice by oral delivery. Recently, several vaccines [23–25,28] and

pharmaceutical peptides [1,32,35] expressed in transgenic

plant seeds are also proved to be orally effective by animal

studies. As described by Walmsley et al., one possible

explanation is that the additional protection against digestion

is afforded through delivery of recombinant peptides in plant

cells (i.e. bioencapsulation within plant cell walls and

membrane compartments) [27]. The increased plasma

rhIGF-1 in oral fed mice implies that transgenic rice seed

system with the BipC fusion strategy in this paper can

overcome barriers and might be a safe and effective oral

delivery system for rhIGF-1. This study indicates the potential

of the novel fusion expression system for the production and

oral delivery of biologically active small peptides for the

medication of various clinical diseases, especially chronic

diseases.

Acknowledgments

This work was supported by a grant of the National ‘863’ High

Technology Program in China (No. 2007AA100505), and the Key

Grant Project of the Chinese Ministry of Education (No.

307018).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.peptides.

2008.07.014.

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